1. Field of the Invention
The present invention relates generally to the fields of nephrology and protein chemistry. More specifically, the present invention relates to the isolation and purification of a novel calcium-independent phospholipase A.sub.2.
2. Description of the Related Art
Phospholipase A.sub.2 (PLA.sub.2) enzymes (EC 3.1.1.4) are a group of lipolytic enzymes that catalyze the hydrolysis of the acyl ester bond at the sn-2 position of glycerophospholipids resulting in the release of arachidonic acid from membrane phospholipids and the production of lysophospholipids (1,2). Mammalian phospholipase A.sub.2 s are structurally a diverse group of enzymes which include the calcium-dependent secreted phospholipase A.sub.2 (sPLA.sub.2) forms characterized by their low molecular weight, high disulfide bond content and an absolute requirement of calcium during hydrolysis (3,4). A second recently identified group of intracellular cytosolic phospholipase A.sub.2 s comprise the low-calcium-requiring 85 kDa cytosolic phospholipase A.sub.2 (cPLA.sub.2) (5,6), and two new calcium-independent phospholipase A.sub.2 enzymes, a 40 kDa plasmalogen selective calcium-independent phospholipase A.sub.2 purified from canine myocardium (7-9) and a 80 kDa cytosolic calcium-independent phospholipase A.sub.2 recently purified from the macrophage-like cell line P388D1 (10-11 ).
In kidney and heart tissue (12-14), early studies have demonstrated that activation of intracellular phospholipase A.sub.2 represents an important mechanism leading to the development of metabolic alterations which precede cell death during ischemia. Recent observations have demonstrated that the majority of measurable phospholipase A.sub.2 activity in freshly isolated rabbit proximal tubules was present in the cytosolic compartment and was calcium-independent and arachidonate-selective (14). These studies also suggest that activation of this calcium-independent phospholipase A.sub.2 enzyme during hypoxia precedes cell death and is accompanied by hydrolysis of endogenous proximal tubule plasmalogen substrates leading to generation of arachidonic acid and accompanying phospholipid catabolism (15). Since the release of arachidonic acid and the concomitant accumulation of amphiphilic products derived from phospholipase A.sub.2 activation such as lysoplasmalogens have been shown to be important modulators of renal NaK-ATPase (16), the isolation and identification of the biochemical mechanisms which regulate the activity of this calcium-independent PLA.sub.2 are of obvious importance.
The prior art is deficient in the lack of the purification and characterization of a novel calcium-independent PLA.sub.2 from rabbit kidney cortex. The present invention fulfills this longstanding need and desire in the art.